Femtosecond to Millisecond Structural Dynamics of A

Home , Femtosecond, Millisecond

Subscriber access provided by HARVARD UNIVERSITY Article Proteins in Action: Femtosecond to Millisecond Structural Dynamics of a Photoactive Flavoprotein Richard James Brust, Andras Lukacs, Allison Laura Haigney, Kiri Addison, Agnieszka Gil, Michael Towrie, Ian P Clark, Gregory M. Greetham, Peter J Tonge, and Stephen Roy Meech J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja407265p • Publication Date (Web): 01 Oct 2013 Downloaded from http://pubs.acs.org on October 7, 2013

Just Accepted

“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties. Page 1 of 9 Journal of the American Chemical Society

1 2 3 4 5 6 7 Proteins in Action: Femtosecond to Millisecond Structural Dynamics 8 9 of a Photoactive Flavoprotein

10 ‡ §,# ‡† § ‡ 11 Richard Brust , Andras Lukacs , Allison Haigney , Kiri Addison , Agnieszka Gil , Michael Tow- 12 rie ║, Ian P. Clark ║, Gregory M. Greetham ║, Peter J. Tonge ‡*, Stephen R. Meech §* 13 ‡ § 14 Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, USA, School of Chemistry, 15 University of East Anglia, Norwich NR4 7TJ, UK, ║Central Laser Facility, Research Complex at Harwell, Harwell Sci- 16 ence and Innovation Campus, Didcot, Oxon OX11 0QX, UK, # Department of Biophysics, Medical School, University 17 of Pecs, Szigeti ut 12, 7624 Pecs, Hungary 18 19 20 ABSTRACT: Living systems are fundamentally dependent on the ability of proteins to respond to external stimuli. The 21 mechanism, the underlying structural dynamics and the time scales for regulation of this response are central questions 22 in biochemistry. Here we probe the structural dynamics of the BLUF domain found in several photoactive flavoproteins 23 where it is responsible for light activated functions as diverse as phototaxis and gene regulation. Measurements have been 24 made over ten decades of time (from 100 fs to 1 ms) using transient vibrational spectroscopy. Chromophore (flavin ring) 25 localized dynamics occur on the pico- to nanosecond timescale, while subsequent protein structural reorganization is 26 observed over microseconds. Multiple time scales are observed for the dynamics associated with different vibrations of 27 the protein, suggesting an underlying hierarchical relaxation pathway. Structural evolution in residues directly H-bonded 28 to the chromophore takes place more slowly than changes in more remote residues. However, a point mutation which 29 suppresses biological function is shown to ‘short circuit’ this structural relaxation pathway, suppressing the changes 30 which occur further away from the chromophore while accelerating dynamics close to it. 31 32 33 INTRODUCTION perturb or even suppress large scale structural changes. 34 Solution phase X-ray scattering has also been applied to 35 The underlying mechanism of protein function involves study PYP dynamics.11,12 Important insights into the shape 36 time dependent changes in structure occurring on multi- 2-4 changes which occur following optical excitation are ob- 37 ple time scales, from sub-picosecond to seconds. Re- tained, but scattering data yield less microscopic struc- 38 cording and modelling the full range of protein dynamics tural detail than diffraction experiments. In this work we 39 is critical to the understanding and manipulation of pro- describe time resolved measurements of light induced 40 tein function. Consequently, the real time measurement structural dynamics in a photoactive flavoprotein in solu- 41 and analysis of protein dynamics is a major objective of tion over a very wide time range, from hundreds of femto- 42 modern biophysics. In many cases, protein activity is seconds to hundreds of microseconds. The protein dy- 43 modulated through interaction with external stimuli such namics are recovered from measurements of the time 44 as allosteric effectors that bind to regions of the protein resolved infra-red difference (TRIR) spectra which are 45 remote from the effector site and result in long range sensitive to structural changes in both the chromophore 46 structural changes. Although allosteric effectors are nor- and the surrounding protein. To achieve this we exploit 47 mally considered to be small organic molecules, photons of light that trigger photoreceptor activation may be con- the recently developed method of ultrafast time resolved 48 14,15 multiple probe spectroscopy (TRMPS). 49 sidered analogous to allosteric modulators. The applica- 50 tion of pulsed lasers to such photoactive proteins thus A number of blue light sensing photoreceptors utilize 51 provides a natural starting time, from which real time flavins as the chromophore, where light absorption is lo- 52 structural dynamics can be measured. For example, time calized in the flavin (isoalloxazine) ring. The photoactive 53 resolved X-ray diffraction has provided detailed insights protein, AppA, is a flavoprotein photoreceptor from into photo-induced structural dynamics in a number of Rhodobacter sphaeroides that regulates photosystem bio- 54 6-13 16,17 55 proteins. The formation of the signalling state of the synthesis in response to both light and oxygen levels. 56 photoactive yellow protein (PYP) has recently been rec- The protein comprises two domains; an N-terminal blue- 57 orded on a one hundred picosecond to millisecond time light utilizing flavin (BLUF) domain, which binds the fla- scale. However, X-ray diffraction requires the protein to vin adenine dinucleotide (FAD) chromophore (Figure 1A), 58 6,8,13 59 be studied in a crystalline environment, which may and a C-terminal domain that is the binding site for the 60 ACS Paragon Plus Environment Journal of the American Chemical Society Page 2 of 9

transcription factor, PpsR. In low light, low oxygen envi- dicative of stronger hydrogen bonding) is observed be- 1 ronments AppA sequesters PpsR, but under blue light tween dark and light adapted states.5,19 Other conserved 2 illumination it undergoes a conformational change result- residues critical to photoactivation include Y21 and 3 ing in the release of the transcription factor, which then W104.29 In the following we report femtosecond to milli- 4 18,19 binds to DNA to inhibit photosystem biosynthesis. second TRIR of dAppA BLUF and two of its mutants (W104A 5 The BLUF domain is of particular interest since it is a and M106A) as they evolve from the dark to the signaling 6 modular unit found in a number of blue light sensing pro- state. We probe both the evolution in protein structure 7 teins where it controls functions as diverse as phototaxis, and the pathway of the structure change as it propagates 8 the photophobic response and gene expression.17,20-22 Re- away from the chromophore. 9 cently it was proposed that the modular nature of the 10 BLUF domain lends itself to applications in the emerging EXPERIMENTAL METHODS 11 field of optogenetics.23 12 Materials. FAD (disodium salt) was from Sigma Aldrich. 13 D2O (99.9 atom %) and [U-13 C6]-D-Glucose (99 atom %) 14 were from Cambridge Isotope Laboratories. Ampicillin 15 (disodium salt), 100x MEM vitamins, and Minimal media 16 were from Fisher. 17 18 Protein Expression and Purification. Mutants were 19 prepared by site directed mutagenesis using pfu Turbo 20 (Agilent). For W104A, the primers used were 5’ TTT GCG 21 GGA GCG CAC ATG CAG CTC TCC TGC TCG 3’ (forward) 22 and 5’ CGA GCA GGA GAG CTG CAT GTG CGC TCC CGC 23 AAA 3’(reverse). For M106A, the primers used were 5’ TTT 24 GCG GGA TGG CAC GCG CAG CTC TCC TGC TCG 3’ 25 (forward) and 5’ CGA GCA GGA GAG CTG CGC GTG CCA TCC CGC AAA 3’ (reverse). AppA BLUF , its mutants and [U- 26 13 27 C]-AppA BLUF were expressed in BL21(DE3) E. coli cells and purified as described previously.28 28 29 Steady State FTIR Spectroscopy. Light minus dark FTIR 30 Figure 1 Structure and H-bonding of FAD in AppA- spectra were obtained on a Vertex 80 (Bruker) FTIR spec- 31 BLUF . (A) Crystal structure of AppA BLUF showing flavin trometer. Here 80 µL of 2 mM protein was placed be- 32 binding between helices 1 and 2 ( B) the hydrogen tween two CaF plates equipped with a 50 µm spacer were 33 bonding network around the flavin that includes the 2 128 scans were accumulated at a 3 cm -1 resolution. The 34 key residues Y21, Q63, W104, and M106. The figure was made using Pymol,1 and the structure 1YRX.pdb.5 (C) light state was generated by 3 minute irradiation using a 35 Details of the proposed H-bonding network changes 460 nm high power mounted LED (Prizmatix). The LED 36 in dAppA BLUF around the chrom ophore. used had a mounted objective providing a focused blue 37 beam on the surface of the infrared cell. 38 BLUF domain proteins exhibit a two state, reversible 39 photocycle characterized by a ca. 10 nm red shift in the TRMPS. The TRMPS method exploits the high signal- 40 absorption spectrum of the flavin ring of FAD, which it- to-noise and stable pulse-to-pulse timing of a 10 kHz am- self remains intact and in its oxidized state in both dark 41 21,22 plified titanium sapphire laser pumping OPAs for the 42 and signaling states. The red shift occurs within 1 ns generation of ~ 50 fs IR probe pulses, described else- and the photocycle is completed by recovery of the dark 14 43 24 where. A second 1 kHz amplified titanium sapphire laser 44 adapted (dAppA) ground state in 30 minutes. A number provides the visible pump pulses (450 nm, ~ 100 fs, 1 µJ, ~ 45 of crystal structures have been solved for the N-terminal 120 µm diameter spot size at the sample). The two ampli- 46 BLUF domain of AppA (AppA BLUF ), revealing an intricate fiers are synchronized with the 65 MHz repetition rate of hydrogen bonding network surrounding the chromo- 47 5,25 the common titanium sapphire seed laser, with the seed 48 phore (Figure 1B). Studies of the structure of dAppABLUF laser optically delayed before the 1 kHz amplifier to 49 and its light adapted signaling state (lAppA BLUF ) suggest achieve 100 fs to 15 ns relative pump – probe delays. For 50 that a key step in forming the signaling state is rotation of delay times between 15 ns and 100 µs the oscillator seed 51 the conserved glutamine (Q63 in AppA) adjacent to the pulse train is used to add steps of 15 ns to the pump laser flavin ring; in-line with this, Q63 is found to be an essen- 52 5,26 delay. For times between 100 µs and 1 ms the 10 kHz probe 53 tial residue for photoactivity. Based on previous work pulses provide a data point every 0.1 ms, until the follow- 54 and our study of the photoinactive mutant Q63E, we pro- ing pump pulse starts the experiment again every 1 ms (at posed a refinement to this model in which tautomeriza- 55 the 1 kHz repetition rate of the pump laser). Pump polari- tion of Q63 precedes rotation, leading to the formation of 56 zation was set to 54.7˚ relative to the IR beam to elimi- a new hydrogen bond to the flavin C4=O carbonyl (Figure 57 nate contributions from orientational relaxation. 1C).27,28 This is consistent with stationary state vibrational 58 spectra, where a red shift in the C4=O stretch mode (in- 59 60 ACS Paragon Plus Environment Page 3 of 9 Journal of the American Chemical Society

Results and Discussion the C-terminal, thus controlling photosystem biosynthe- 1 Chromophore Dynamics Figure 2A shows the time sis. This requires analysis of the TRIR spectra beyond 10 2 dependent TRIR spectra for dAppA between 2 ps and ns. 3 BLUF 10 ns after 450 nm excitation of the flavin ring of FAD, the Protein Dynamics Although it is evident from Figure 4 chromophore responsible for blue light absorption in 2A that most population has returned to the ground state 5 photoactive flavoproteins. These difference spectra com- within 10 ns, a weak spectrum remains; it is the evolution 6 prise: negative bands (bleaches) associated with depletion of this spectrum which reveals the subsequent protein 7 of the flavin ground state population, or with photo- dynamics. Exploiting an apparatus with high signal-to- 8 induced changes in the vibrational spectrum of the pro- noise, a 10 kHz data acquisition rate and the ability to 9 15 tein, occurring either directly through electronic excita- measure TRIR from 100 fs to 1 ms, we have time resolved 10 tion at t = 0 or as a result of subsequent structural dynam- the data beyond 10 ns (Figure 2B). It was established (Fig- 11 ics; positive bands associated with vibrations of the elec- ure 2A) that by 10 ns the initially excited singlet state of 12 tronically excited state of the flavin, or with modes of the FAD has completely relaxed (e.g. from the disappearance 13 -1 protein which shift as a result of electronic excitation, or of the 1383 cm transient mode of the flavin S state). 14 1 of products formed subsequently. The dominant sub- However, the ground state has not been completely re- 15 nanosecond relaxation is well fit by a biexponential func- populated as can be seen from the persistence of flavin 16 tion with components of tens and hundreds of picose- localized bleach modes at 1547 and 1700 cm -1 in the TRIR. 17 conds consistent with an inhomogeneous distribution of In addition a weak transient, possibly a triplet state (See 18 ground state structures leading to a distribution of decay supporting information, Figure S1), is formed, giving rise 19 rates (Supporting Information Table S1).27,28,30 The two to the weak absorption near 1440 cm -1. The observation of 20 highest frequency bleach modes at 1700 and 1650 cm -1 are residual ground state bleaches after the excited state de- 21 associated with two carbonyl stretches of the FAD ground cay is complete proves the existence of long-lived inter- 22 state, and are sensitive to the H-bond environment.31-34 mediate protein structures in the photocycle. Figure 2B 23 The intense bleach at 1547 cm -1 and the weaker one at probes the relaxation of this structure on a longer time- 24 1580 cm -1 are FAD ring modes. The two positive peaks scale, and shows that complete recovery of the 1547 cm -1 25 formed immediately on excitation at 1408 cm -1 and 1383 chromophore ground state mode occurs on the microsec- 26 cm -1 are not assigned to specific vibrational modes, but ond timescale, while the mode associated with the C4=O 27 are associated with the excited state of the chromophore carbonyl of the flavin ring at 1700 cm -1 recovers with the 28 rather than with frequency shifted protein modes, as same rate (Table I), but to a non-zero bleach level, i.e. this 29 proven by comparison with the TRIR of the flavin ring in chromophore mode has not fully recovered its initial state 30 free solution (Supporting Information Figure S1). within 50 µs. Data beyond 50 µs (out to 1 ms) showed no 31 further change in the TRIR spectrum. These data thus 32 The mechanism of the primary photochemical step in AppA underlying the data of Figure 2A is controversial. point to microsecond dynamics refilling the original 33 ground state, while the latter feature indicates that the 34 Ultrafast optical spectroscopy assigned the primary step to electron transfer between excited FAD and an adjacent spectrum associated with the signaling state, a shifted 35 19 conserved tyrosine residue, Y21 (Figure 1B) followed by flavin C4=O mode due to altered H-bond interactions, 36 proton transfer.24,35-37 The resultant changes in electronic has formed within tens of microseconds. 37 structure were proposed to lead to changes in the H- Most significantly, microsecond dynamics associated 38 bonding network prior to back electron transfer. This specifically with the protein are evident in the complex 39 assignment was based on analysis of the complex multi- dispersive band profile between 1600 and 1640 cm -1, which 40 phase kinetics and observations of a radical-like spectrum also continues to evolve after the excited state decay of 41 in the transient visible absorption of a related BLUF do- FAD (Figure 2B). Since there are no strong flavin chromo- 42 main protein, PixD.30 We proposed an alternative mecha- phore modes in this region (supporting information, Fig- 43 nism for AppA photoactivity where excitation of FAD ure S1), these changes must be assigned to structural evo- 44 itself is sufficient to induce a change in the H-bonding lution in the protein. This result demonstrates the sensi- 45 network, giving rise to keto-enol tautomerization in Q63, tivity of vibrational spectroscopy to protein dynamics; in 46 which then leads on to the required structure change. the electronic spectrum no evolution was detected be- 47 This assignment was based on the absence of radical tween 10 ns and 15 µs.41 The assignment of the 1622/1631 48 -1 states of FAD in the sub-nanosecond TRIR spectra of cm dispersive profile to protein modes was confirmed by 49 27 13 dAppA BLUF , the observed prompt perturbation of the repeating the experiment in uniformly C labeled protein, 50 13 protein network on excitation and the relative quenching U- C dAppA BLUF ; TRIR data recorded after 10 ns and 20 µs 51 27,28 and recovery kinetics of FAD. There is theoretical sup- are shown in Figure 2C. Both parts of the dispersive line- 52 38-40 -1 port for both mechanisms, and, importantly, both shape are red-shifted by 36 ± 2 cm from the unlabeled 53 agree that light induced structure change in the network spectrum, consistent with an isotope shift. In contrast, 54 28 of amino acids surrounding FAD occurs within 1 ns, which modes assigned to the FAD chromophore are unshifted. 55 ultimately leads to formation of the signaling state. The assignment of the dispersive band to protein is in 56 good agreement with the stationary state IR difference 57 The focus of this paper is on the previously hidden evolution in protein structure which propagates from the N- spectra of Masuda and co-workers, who proposed on the 58 basis of the observed 59 terminal FAD binding site to ultimately release PpsR at 60 ACS Paragon Plus Environment Journal of the American Chemical Society Page 4 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Figure 2. Time resolved IR difference spectra for dAppA BLUF . (A) TRIR spectra recorded between 2 ps and 10 ns after 29 excitation of dAppA BLUF at 450 nm. The fast and complete decay of the singlet excited state is evident in the transient flavin 30 modes at 1380 cm -1. However, the ground state recovery is incomplete e.g. at 1547 cm -1 and some transient (probably triplet) 31 state is formed. ( B) Relaxation in the dAppA BLUF TRIR spectrum between 10 ns and 50 µs after excitation. The electronic 32 ground state recovers fully (1547 cm -1) but formation of a new environment is indicated by the shift and incomplete recovery 33 in the carbonyl mode at 1703 cm -1. The temporal evolution in the 1622/1631 cm -1 pair of protein modes is also evident. ( C) 13 34 Effect of C isotope exchange in dAppA BLUF measured 10 ns and 20 µs after excitation ( D) Comparison of the TRIR spectra 35 recorded 20 µs after excitation with the stationary state IR difference spectrum for the light minus dark states. 36 frequencies that these changes arose from a C=O (amide) Further detail can be recovered from analysis of the ki- 37 mode of the β-sheet structure, which is linked to FAD netics between 10 ns and 10 µs after excitation. For the 38 bound in the α-helix region by the key residues W104, 1622/1631 cm -1 dispersive pair (associated with protein 39 Q63 and Y21 (Figure 1B).19 bleach and absorption respectively) the kinetics are pre- 40 In figure 2D the TRIR spectrum recorded at 20 µs is sented in Figure 3A. A striking result is that these two 41 compared with a steady state IR difference spectrum rec- bands are kinetically distinct and not therefore linked by 42 -1 orded with the same (approximately 3 cm ) resolution. a simple first order shift to lower frequency of a single 43 -1 These spectra show that the structural dynamics associat- protein mode. The transient absorption at 1631 cm rises 44 ed with these protein modes are essentially complete in ca. 1.5 µs, which is reproducibly faster than the 2.1 µs 45 -1 within 20 µs, with no further changes in TRIR being ob- development of the 1622 cm bleach (Table I). This result 46 served out to 1 ms. The formation of the signaling state is not unexpected, as the structural changes between the 47 within 20 µs is however longer than the nanosecond time- light and dark states are spread over a number of resi- 48 42 scale associated with the red shift of the FAD chromo- dues, each of which may have a slightly different vibra- 49 41 phore observed by ultrafast electronic spectroscopy. The tional frequency associated with its amide backbone. The 50 microsecond timescale is significant in the light of an kinetics associated with changes occurring on more than 51 NMR study of light and dark adapted forms of the BLUF one residue will be both more complex than a simple first 52 domain, which suggested that the structural changes order process and hierarchical in nature. This is likely to 53 which occur are of small scale but take place in residues result in stretched exponential or dispersive kinetics ra- 43 54 relatively remote from the FAD chromophore, including ther than single exponential relaxation, although the 55 in the β-sheet.42 The present data thus show that struc- present signal-to-noise does not permit the extraction of 56 tural changes taking place at distances in excess of 10 Å anything more than a characteristic timescale for the dy- 57 from the chromophore can occur on the microsecond namics associated with each mode. 58 timescale. 59 60 ACS Paragon Plus Environment Page 5 of 9 Journal of the American Chemical Society

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Figure 3. Comparison of protein and chromophore mode kinetics. (A) Kinetics of protein modes, showing that the 17 linked pair at 1622/1631 cm -1 exhibit distinct kinetics. The growth of the transient occurs more rapidly than the evolution of 18 the bleach. ( B) Kinetics associated with the recovery of the chromophore modes at 1547 cm -1 (complete recovery) and 1703 19 cm -1 (partial recovery (Figure 2B)) and the growth of the 1688 cm -1 transient. The slower dynamics associated with the chro- 20 mophore recovery and growth of the light adapted state compared to the protein modes in ( A) is apparent. The relevant 21 optical density axes are indicated by the symbol color. 22 23 Consideration of the relaxation times of other modes is larities in timescales mean that we cannot rule out a role 24 consistent with this more complex picture (Figure 3B). for the triplet state in the microsecond recovery kinetics 25 The weak transient feature at 1688 cm -1 develops on a of the BLUF domain ground electronic state at 1547 cm -1. 26 longer timescale than the protein modes (Table I), but on We complemented the above analysis of individual as- 27 the same timescale as the partial bleach recovery at 1703 signed modes with a global analysis of the ps –µs spec- 28 cm -1. The 1688 cm -1 feature is well resolved in the light trum (Supporting Information Figure S3), assuming a se- 29 minus dark difference spectrum (Figure 2D) and was as- quence of first order kinetics, which reproduced the be- 30 signed previously by Masuda and co-workers to an in- havior described here. 31 crease in H-bonding between the flavin carbonyl mode Modification of Protein Dynamics in W104A AppA- 32 and protein in the light adapted state. The present results BLUF To further characterize the relaxation pathway in 33 thus suggest a phase in protein structural reorganization AppA BLUF we measured the 100 fs to 1 ms dynamics associ- 34 slower than seen in the protein modes in Figure 3A. Sig- ated with two mutants which both link the flavin binding 35 nificantly, the slower structural reorganization occurs in pocket to the β-sheet (Figure 1A) and show the red-shift 36 protein residues sufficiently close to the flavin chromo- in FAD absorption between dark and light adapted states 37 phore to be involved in H-bonding with it. This contrasts characteristic of photoactivity: W104A and M106A. Com- 38 with the faster changes assigned to the more remote resi- pared to dAppA BLUF , W104A is known to dramatically ac- 39 dues in the β-sheet (Figure 3A). This result shows that celerate the recovery of the dark state, by a factor of 80, 40 there is no simple relationship between the timescale of while M106A gives rise to a more modest factor of 1.5.44 41 the protein response to electronic excitation and distance There are distinct differences between the TRIR data for 42 from the chromophore. W104A and dAppA BLUF between 100 fs to 10 ns (Figures 2A 43 The chromophore bleach modes (e.g. at 1547 cm -1) also and 4A). Most strikingly the flavin ground state (1547 cm - 44 recover on this longer timescale, as does the very weak 1) recovers and a red-shifted C4=O transient species (1688 45 transient absorption around 1440 cm -1. These might both cm -1) develops simultaneously, both on the nanosecond 46 be assigned to relaxation of a triplet state, since a similar timescale (Figure 4A and S3). The 1688 cm -1 mode is as- 47 (but faster) evolution is observed for FMN in aqueous signed to a rearrangement in H-bonding between the 48 solution (Figure S1). In an effort to resolve the triplet state protein and the flavin ring 19 and only appears on the mi-

49 in AppA BLUF we studied the photoinactive mutant Q63E crosecond timescale in dAppA BLUF (Figure 3); evidently 50 on the picosecond to microsecond timescale (Figure S2).28 this rearrangement has very different dynamics in W104A. 51 In this case no 1440 cm -1 transient was observed. However, A second striking difference is in the kinetics associated 52 the recovery of the flavin ground state was markedly fast- with the protein mode lineshape on the nanosecond to

53 er in Q63E than in dAppA BLUF (Figure S2), so this result millisecond time scale (Figure 4B). In W104A the positive 54 cannot rule out a contribution from the triplet state. feature (1631 cm -1) appears immediately and shows no 55 Thus, although the developing transient absorption at further evolution, while the negative feature (1622 cm -1) 56 1688 cm -1 can be reliably assigned to a change in the pro- does grow over time but to a level which is much weaker

57 tein environment around the chromophore which is than in dAppA BLUF . This is clearly illustrated by the com- 58 slower than the changes seen at 1622/1631 cm -1, the simi- parison of the transient spectra for W104A, 59 60 ACS Paragon Plus Environment Journal of the American Chemical Society Page 6 of 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 Figure 4 Transient IR spectra for dAppA BLUF and two mutants. (A) Femtosecond to nanosecond TRIR of W104A. ( B) 30 Microsecond dynamics of W104A. ( C) Comparison of the TRIR spectra of AppABLUF and the two mutants 10 ns after excita- 31 tion. ( D) As for ( C) but 20 µs after excitation. 32

33 34 M106A and dAppA BLUF at 10 ns and 10 µs (Figures 4C and the microsecond TRIR of dAppA BLUF is ‘short circuited’ i.e. 35 D respectively). In Figure 4C the fast appearance of the there is a light induced change in the local H-bonding -1 36 1688 and 1631 cm features is apparent, while Figure 4D environment of the FAD chromophore, which leads to the 37 shows that there is much weaker development of the nanosecond spectral shift in the C4=O mode, but that the -1 38 transient bleach (1622 cm ) in W104A, while dAppA BLUF longer range structural changes observed in dAppA BLUF , and M106A are very similar, and in particular both show and critical for protein function, do not develop. Instead, 39 -1 40 the development of the 1688 cm transient and the pro- the ground state structure recovers on a time scale similar 41 tein modes occur on the microsecond timescale (Table 1, to that of dAppA BLUF (Table I). 42 Figure S5). Inspection of the kinetics associated with each Such a short range change in structure is consistent 43 mode (Figure S5) confirms the lack of development be- with the eighty-fold increase in the rate of dark state re- yond 10 ns for most modes in W104A and shows that the covery in W104A, compared to dAppA and with bio- 44 -1 BLUF 45 1622 cm bleach mode develops more rapidly than in chemical measurements of AppA antirepressor activity, 46 M106A and dAppA BLUF , which are in every respect similar. 47 These data confirm that W104 is a key residue in com- Table 1. Kinetic analysis of AppA BLUF , W104A and 48 municating the electronic excitation of the flavin ring to M106A. 49 the protein backbone.45 It was established in steady state Peak/ cm -1 dAppA /µs W104A/µs M106A/µs 50 IR difference measurements that mutations in W104 sup- BLUF 46 51 press the appearance of protein modes. The present data 1547 5.4 ± 0.5 5.2 ± 0.6 4.5 ± 0.5 52 shows that this is a mechanistic change rather than a ki- 53 netic one, i.e. for W104A the photoinduced change in pro- 1622 2.1 ± 0.3 2.6 ± 0.6 2.2 ± 0.4 54 tein structure observed in dAppA BLUF never occurs, rather 1631 1.5 ± 0.3 N.A. 1.2 ± 0.4 55 than occurs and rapidly relaxes. It is significant that 56 W104A forms the red shifted flavin carbonyl associated 1688 5.6 ± 0.8 N.A. 6.3 ± 1.1 57 with the signaling state (Figure 4), and that this mode 1703 5.3 ± 0.7 N.A. 5.8 ± 0.8 58 forms on the nanosecond time scale (Figures 4A, S3). We 59 suggest that in W104A the structural evolution revealed in 60 ACS Paragon Plus Environment Page 7 of 9 Journal of the American Chemical Society

which showed much lower activity for W104A than for ACKNOWLEDGMENT 1 wild-type.29 Evidently, a spectral shift alone is not suffi- 2 cient to indicate a photoactive state of the BLUF domain. The authors are grateful to STFC for access to the central 3 laser facility. RB and AG thank the OPPF for their assistance

4 in sample preparation. KA thanks UEA for the award of a 5 Conclusions studentship. 6 Time resolved infra-red spectroscopy has revealed the 7 timescale and pathway of structural dynamics in a BLUF ABBREVIATIONS 8 domain. Structural dynamics were probed on the 100 fs to FAD, flavin adenosine dinucleotide; BLUF, blue light sensing 9 1 ms time scale. Following electronic excitation, the pri- using FAD; AppA, activation of photopigment and PUC A 10 mary events are associated with relaxation of the flavin protein; FTIR, Fourier Transform infrared; TRIR, time re- 11 excited electronic state. This occurs on a sub-nanosecond solved infrared; TRMPS, time resolved multiple probe spec- 12 timescale, and mainly results in recovery of the initial troscopy. 13 ground state, with a minor fraction of excited states lead- 14 ing to perturbation of the local structure of the protein REFERENCES 15 and possibly some triplet state formation. The secondary (1) The Pymol Molecular Graphics System, 16 steps involve protein structural dynamics which convert Version 1.5.0.4. Shrödinger, LLC (2) Agarwal, P. K.; Billeter, S. R.; Rajagopalan, P. 17 these local perturbations into the changes which eventu- T. R.; Benkovic, S. J.; Hammes-Schiffer, S. Proc. Natl. Acad. Sci. ally form the signaling state, releasing the repressor mole- 18 U. S. A. 2002 , 99 , 2794. 19 cule. The time scale for these protein structural dynamics (3) Yang, H.; Luo, G. B.; Karnchanaphanurach, 20 is 1-5 µs. The fastest steps communicate optical excitation P.; Louie, T. M.; Rech, I.; Cova, S.; Xun, L. Y.; Xie, X. S. Science 21 to the protein via the W104 residue in 1-2 µs, and more 2003 , 302 , 262. 22 than one residue is involved, such that different protein (4) Lange, O. F.; Lakomek, N. A.; Fares, C.; 23 modes present different response times. There are also Schroder, G. F.; Walter, K. F. A.; Becker, S.; Meiler, J.; 24 slower dynamics which reflect relaxation in the vicinity of Grubmuller, H.; Griesinger, C.; de Groot, B. L. Science 2008 , 320 , 25 the chromophore, showing that the rate of structural 1471. (5) Anderson, S.; Dragnea, V.; Masuda, S.; Ybe, change is not simply related to distance from the chro- 26 J.; Moffat, K.; Bauer, C. Biochemistry 2005 , 44 , 7998. 27 mophore. In the W104A mutant communication to the (6) Cho, H. S.; Dashdorj, N.; Schotte, F.; Graber, 28 protein is suppressed, but fast H-bond rearrangements T.; Henning, R.; Anfinrud, P. Proc. Natl. Acad. Sci. U. S. A. 2010 , 29 still occur around the chromophore, suggesting changes 107 , 7281. 30 in the chromophore spectrum are not a good measure of (7) Ihee, H.; Rajagopal, S.; Srajer, V.; Pahl, R.; 31 photoactivity. Anderson, S.; Schmidt, M.; Schotte, F.; Anfinrud, P. A.; Wulff, 32 M.; Moffat, K. Proc. Natl. Acad. Sci. U. S. A. 2005 , 102 , 7145. 33 ASSOCIATED CONTENT (8) Schotte, F.; Cho, H. S.; Kaila, V. R. I.; Kamikubo, H.; Dashdorj, N.; Henry, E. R.; Graber, T. J.; Henning, (Supporting Information . Transient IR spectra of FMN in 34 R.; Wulff, M.; Hummer, G.; Kataoka, M.; Anfinrud, P. A. Proc. 35 solution and Q63E are provided in supporting information Natl. Acad. Sci. U. S. A. 2012 , 109 , 19256. 36 along with some further kinetic data. This material is availa- (9) Ihee, H.; Lorenc, M.; Kim, T. K.; Kong, Q. Y.; 37 ble free of charge via the Internet at http://pubs.acs.org. Cammarata, M.; Lee, J. H.; Bratos, S.; Wulff, M. Science 2005 , 38 309 , 1223. 39 ASSOCIATED CONTENT (10) Kim, K. H.; Muniyappan, S.; Oang, K. Y.; Kim, J. G.; Nozawa, S.; Sato, T.; Koshihara, S. Y.; Henning, R.; 40 AUTHOR INFORMATION 41 Kosheleva, I.; Ki, H.; Kim, Y.; Kim, T. W.; Kim, J.; Adachi, S.; Ihee, H. Journal of the American Chemical Society 2012 , 134 , 7001. 42 Corresponding Author (11) Kim, T. W.; Lee, J. H.; Choi, J.; Kim, K. H.; 43 *PJT: Telephone: (631) 632-7907; Fax: (631) 632-7960; Email: van Wilderen, L. J.; Guerin, L.; Kim, Y.; Jung, Y. O.; Yang, C.; 44 [email protected]; *SRM:44(0)1603 593141; Fax: Kim, J.; Wulff, M.; van Thor, J. J.; Ihee, H. Journal of the American 45 44(0)1603 592004; Email: [email protected] Chemical Society 2012 , 134 , 3145. 46 (12) Ramachandran, P. L.; Lovett, J. E.; Carl, P. J.; 47 Present Addresses Cammarata, M.; Lee, J. H.; Jung, Y. O.; Ihee, H.; Timmel, C. R.; 48 † The Wistar Institute, Philadelphia, PA 19104, USA . van Thor, J. J. Journal of the American Chemical Society 2011 , 133 , 49 9395. Author Contributions (13) Jung, Y. O.; Lee, J. H.; Kim, J.; Schmidt, M.; 50 The manuscript was written through contributions of all Moffat, K.; Šrajer, V.; Ihee, H. Nat Chem 2013 , 5, 212. 51 authors. All authors have given approval to the final version (14) Greetham, G. M.; Burgos, P.; Cao, Q. A.; 52 of the manuscript. Clark, I. P.; Codd, P. S.; Farrow, R. C.; George, M. W.; Kogimtzis, 53 M.; Matousek, P.; Parker, A. W.; Pollard, M. R.; Robinson, D. A.; 54 Funding Sources Xin, Z. J.; Towrie, M. Applied Spectroscopy 2011 , 64 , 1311. 55 Funded by EPSRC (EP/K000764/1) o SRM), STFC (pro- (15) Greetham, G. M.; Sole, D.; Clark, I. P.; Parker, 56 gramme 101005 to SRM and PJT) and NSF (CHE-1223819 to A. W.; Pollard, M. R.; Towrie, M. Rev. Sci. Instrum. 2012 , 83 . (16) Gomelsky, M.; Klug, G. Trends in Biochemical 57 PJT). Sciences 2002 , 27 , 497. 58 59 60 ACS Paragon Plus Environment Journal of the American Chemical Society Page 8 of 9

(17) Losi, A.; Gartner, W. Photochemistry and (39) Nunthaboot, N.; Tanaka, F.; Kokpol, S. 1 Photobiology 2010 , 87 , 491. Journal of Photochemistry and Photobiology a-Chemistry 2009 , 2 (18) Willis, V. C.; Gizinski, A. M.; Banda, N. K.; 207 , 274. 3 Causey, C. P.; Knuckley, B.; Cordova, K. N.; Luo, Y.; Levitt, B.; (40) Nunthaboot, N.; Tanaka, F.; Kokpol, S. 4 Glogowska, M.; Chandra, P.; Kulik, L.; Robinson, W. H.; Arend, Journal of Photochemistry and Photobiology a-Chemistry 2010 , 5 W. P.; Thompson, P. R.; Holers, V. M. J Immunol 2011 , 186 , 4396. 209 , 79. (19) Masuda, S.; Hasegawa, K.; Ono, T. (41) Gauden, M.; Yeremenko, S.; Laan, W.; van 6 Biochemistry 2005 , 44 , 1215. Stokkum, I. H. M.; Ihalainen, J. A.; van Grondelle, R.; 7 (20) Ito, S.; Murakami, A.; Sato, K.; Nishina, Y.; Hellingwerf, K. J.; Kennis, J. T. M. Biochemistry 2005 , 44 , 3653. 8 Shiga, K.; Takahashi, T.; Higashi, S.; Iseki, M.; Watanabe, M. (42) Wu, Q.; Ko, W. H.; Gardner, K. H. 9 Photochemical & Photobiological Sciences 2005 , 4, 762. Biochemistry 2008 , 47 , 10271. 10 (21) Masuda, S.; Bauer, C. E. Cell 2002 , 110 , 613. (43) Osvath, S.; Herenyi, L.; Zavodszky, P.; Fidy, 11 (22) Masuda, S. Plant and Cell Physiology 2013 , 54 , J.; Kohler, G. Journal of Biological Chemistry 2006 , 281 , 24375. 12 171. (44) Dragnea, V.; Arunkumar, A. I.; Yuan, H.; 13 (23) Stierl, M.; Stumpf, P.; Udwari, D.; Gueta, R.; Giedroc, D. P.; Bauer, C. E. Biochemistry 2009 , 48 , 9969. Hagedorn, R.; Losi, A.; Gartner, W.; Petereit, L.; Efetova, M.; (45) Masuda, S.; Hasegawa, K.; Ono, T. A. Plant 14 Schwarzel, M.; Oertner, T. G.; Nagel, G.; Hegemann, P. Journal of and Cell Physiology 2005 , 46 , 1894. 15 Biological Chemistry 2011 , 286 , 1181. (46) Hasegawa, K.; Masuda, S.; Ono, T. A. Plant 16 (24) Laan, W.; Gauden, M.; Yeremenko, S.; van and Cell Physiology 2005 , 46 , 136. 17 Grondelle, R.; Kennis, J. T. M.; Hellingwerf, K. J. Biochemistry 18 2006 , 45 , 51. 19 (25) Jung, A.; Domratcheva, T.; Tarutina, M.; Wu, 20 Q.; Ko, W. H.; Shoeman, R. L.; Gomelsky, M.; Gardner, K. H.; 21 Schlichting, L. Proc. Natl. Acad. Sci. U. S. A. 2005 , 102 , 12350. (26) Grinstead, J. S.; Hsu, S. T. D.; Laan, W.; 22 Bonvin, A.; Hellingwerf, K. J.; Boelens, R.; Kaptein, R. 23 Chembiochem 2006 , 7, 187. 24 (27) Stelling, A. L.; Ronayne, K. L.; Nappa, J.; 25 Tonge, P. J.; Meech, S. R. Journal of the American Chemical 26 Society 2007 , 129 , 15556. 27 (28) Lukacs, A.; Haigney, A.; Brust, R.; Zhao, R. K.; 28 Stelling, A. L.; Clark, I. P.; Towrie, M.; Greetham, G. M.; Meech, 29 S. R.; Tonge, P. J. Journal of the American Chemical Society 2011 , 133 , 16893. 30 (29) Masuda, S.; Tomida, Y.; Ohta, H.; Takamiya, 31 K. I. Journal of Molecular Biology 2007 , 368 , 1223. 32 (30) Bonetti, C.; Stierl, M.; Mathes, T.; van 33 Stokkum, I. H. M.; Mullen, K. M.; Cohen-Stuart, T. A.; van 34 Grondelle, R.; Hegemann, P.; Kennis, J. T. M. Biochemistry 2009 , 35 48 , 11458. 36 (31) Haigney, A.; Lukacs, A.; Brust, R.; Zhao, R. K.; 37 Towrie, M.; Greetham, G. M.; Clark, I.; Illarionov, B.; Bacher, A.; Kim, R. R.; Fischer, M.; Meech, S. R.; Tonge, P. J. Journal of 38 Physical Chemistry B 2012 , 116 , 10722. 39 (32) Haigney, A.; Lukacs, A.; Zhao, R. K.; Stelling, 40 A. L.; Brust, R.; Kim, R. R.; Kondo, M.; Clark, I.; Towrie, M.; 41 Greetham, G. M.; Illarionov, B.; Bacher, A.; Romisch-Margl, W.; 42 Fischer, M.; Meech, S. R.; Tonge, P. J. Biochemistry 2011 , 50 , 1321. 43 (33) Kondo, M.; Nappa, J.; Ronayne, K. L.; 44 Stelling, A. L.; Tonge, P. J.; Meech, S. R. Journal of Physical 45 Chemistry B 2006 , 110 , 20107. (34) Lukacs, A.; Zhao, R. K.; Haigney, A.; Brust, R.; 46 Greetham, G. M.; Towrie, M.; Tonge, P. J.; Meech, S. R. Journal of 47 Physical Chemistry B 2012 , 116 , 5810. 48 (35) Alexandre, M. T. A.; van Wilderen, L. J. G.; 49 van Grondelle, R.; Hellingwerf, K. J.; Groot, M. L.; Kennis, J. T. M. 50 Biophysical Journal 2005 , 88 , 509A. 51 (36) Gauden, M.; Grinstead, J. S.; Laan, W.; van 52 Stokkum, I. H. M.; Avila-Perez, M.; Toh, K. C.; Boelens, R.; 53 Kaptein, R.; van Grondelle, R.; Hellingwerf, K. J.; Kennis, J. T. M. Biochemistry 2007 , 46 , 7405. 54 (37) Mathes, T.; van Stokkum, I. H. M.; Bonetti, 55 C.; Hegemann, P.; Kennis, J. T. M. Journal of Physical Chemistry 56 B 2011 , 115 , 7963. 57 (38) Domratcheva, T.; Grigorenko, B. L.; 58 Schlichting, I.; Nemukhin, A. V. Biophysical Journal 2008 , 94 , 59 3872. 60 ACS Paragon Plus Environment Page 9 of 9 Journal of the American Chemical Society

1 SYNOPSIS TOC (Word Style “SN_Synopsis_TOC”). 2 3 Authors are required to submit a graphic entry for the Table of Contents (TOC) that, in conjunction with the man- 4 uscript title, should give the reader a representative idea of one of the following: A key structure, reaction, equation, 5 concept, or theorem, etc., that is discussed in the manuscript. Consult the journal’s Instructions for Authors for 6 TOC graphic specifications. 7 8 9 10 11 12 13 14 15 16 Insert Table of Contents artwork here 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment